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Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus
Journal of Invertebrate Pathology 134 (2016) 48–53
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Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus Chen Li, Xiao-Xiao Gao, Jie Huang, Yan Liang ⇑ Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
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Article history: Received 29 September 2015 Revised 9 January 2016 Accepted 12 January 2016 Available online 13 January 2016 Keywords: WSSV Shrimp F1 ATP synthase b subunit BP53 VP37 Interaction
a b s t r a c t The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase b subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson’s correlation coefficients reached to 0.67 ± 0.05 and the Mander’s overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. Ó 2016 Elsevier Inc. All rights reserved.
0. Introduction For a virus to initiate its life cycle, specific binding must occur between viral attachment proteins (VAPs) and cellular receptors on host cells, which mediates virus entry and triggers subsequent viral infection (Boyle et al., 1987). Associations between VAPs and host receptor molecules can occur via high- or low-affinity interactions, and are thought to be important for cell fusion and viral replication (Staunton et al., 1989; Compton et al., 1993). The shrimp farming industry is affected by several viral diseases that cause billions of dollars of economic losses annually (Lightner, 1996). White spot syndrome virus (WSSV, family Nimaviridae, genus Whispovirus), an enveloped, double-stranded DNA virus, containing 180 putative open reading frames (ORFs) in its complete genome sequence (Tsai et al., 2000; Van et al., 2001; Yang et al., 2001), is a severe viral pathogen (Lightner, 1996). Over 50 structural and several non-structural protein genes of WSSV have ⇑ Corresponding author at: No. 106 Nanjing Road, Qingdao 266071, China. E-mail address: [email protected] (Y. Liang). http://dx.doi.org/10.1016/j.jip.2016.01.006 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
been characterized, including VP26, VP28, VP68, VP187, and VP466 that have been reported to be significantly involved in WSSV infection (Xie and Yang, 2005; Wu et al., 2005; Sritunyalucksana et al., 2006; Ye et al., 2012). Previously, we suggested that the envelope protein WSSV-VP37 is a viral attachment protein and can interact with shrimp cells (Liang et al., 2005; Liu et al., 2006; Liu et al., 2009). Virus-host cell interactions play an important role in WSSV infection, as summarized by Sritunyalucksana et al. (2012). Using a virus overlay protein binding assay (VOPBA) with WSSV and in vivo neutralization tests, we found that shrimp F1 ATP synthase b subunit (ATPsyn b, termed BP53) plays a role in WSSV infection (Liang et al., 2010). Further studies of BP53 revealed its location on the cell surface of shrimp hemocytes and gill epithelial cells, which meet the basic requirements for it to be a viral receptor (Zhan et al., 2013). In our previous study, we showed evidence that ATP synthesis is active on the shrimp cell surface and can be suppressed by WSSV infection (Y. Liang et al., 2015). As ATP synthase b – a part of the F1Fo ATP synthase complex – can serve as a receptor for WSSV, our findings support the hypothesis that WSSV disturbs host energy
C. Li et al. / Journal of Invertebrate Pathology 134 (2016) 48–53
metabolism by interacting with host BP53 molecules (Y. Liang et al., 2015). Recently, BP53 was reported to be a VP37-binding protein (Zhan et al., 2013). However it’s unclear how many viral proteins, excluding VP37, could interact with BP53. There also lacks detailed description on the relationship between shrimp BP53 and WSSV-VP37. This study aimed to identify and assess interactions between BP53 and its viral binding proteins by both qualitative and quantitative analyses. Investigating the relationship between shrimp BP53 and viral binding proteins might represent an interesting way to better understand the molecular pathways involved in WSSV infection.
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to react with viral proteins, and was incubated with the eluted products at 37 °C for 2 h. After three washes with PBS containing 0.05% Tween 20, membranes were incubated with 1:5000 rabbit anti-rBP53 polyclonal antibody at 37 °C for 2 h. The WSSV viral proteins on the other membrane were incubated with either 1:5000 rabbit anti-rVP37 polyclonal antibody or rabbit antirVP28 polyclonal antibody at 37 °C for 2 h. After three times wash, the signal was detected using a HRP-conjugated goat anti-rabbit IgG secondary antibody. After thorough washing, the color was developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., USA). 1.5. Determination of binding capability between VP37 and BP53
1. Materials and methods 1.1. Virus source Intact WSSV viral particles from infected crayfish tissues were purified by differential centrifugation as described by Xie et al. (2005). Viral protein concentrations were measured using the Bradford method (Stoscheck, 1990) and proteins were stored at 80 °C. 1.2. Recombinant VP37 and BP53 Recombinant VP37 (rVP37) and recombinant BP53 (rBP53) were produced in an Escherichia coli expression system, as reported by Liu et al. (2009) and Liang et al. (2010), respectively. In brief, TOP10s encoding recombinant plasmid pBAD/gIII A-VP37 were detected by PCR, and 16S rDNA was used as a reference gene. Primers designed using Primer Premier 5.0 showed in Table 1. The rVP37 was purified using HisTrap HF resin and GE AKTA Explorer 100 (GE Inc., USA) according to the manufacturer’s instructions under denatured conditions, and then was renatured by successive for 12 h incubations with 6, 4, 2, and 0 M Guanidine–HCl in buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 25 mM dithiothreitol, 0.1% Tween-20, and 10% glycerol, pH 7.5) at 4 °C. Fractions were collected using the Centrifugal Filters (Millipore Inc., USA) and then analyzed by SDS–PAGE. 1.3. Polyclonal antibodies Rabbit polyclonal antibodies against VP37 and VP28 were custom prepared by GenScript (Nanjing Co. in China) by immunizing New Zealand white rabbits with either purified antigenic protein rVP37 or rVP28. The rabbit polyclonal antibody against BP53 was prepared as reported by Y. Liang et al. (2015). Antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). 1.4. Determination of binding proteins for BP53 Far-western blotting assays were carried out to identify binding proteins for BP53. The WSSV proteins were separated by 12% SDS– PAGE and transferred to a membrane. After blocking with 5% skim milk in PBS at 37 °C for 1 h, purified rBP53 was applied as a probe Table 1 Primers for pBAD/gIII A-VP37 and 16SrDNA.
To determine the binding capability of rVP37 and WSSV viral proteins with rBP53, an ELISA binding assay was done. Along with a similar amount of protein (30 lg) in skim milk, rVP37 and WSSV viral proteins were coated in flat-bottomed 96-well plates (Corning, China) at 4 °C overnight and then were blocked with 5% nonfat milk in PBS buffer for 2 h at 37 °C. Plates were washed three times with PBS buffer that contained 0.05% Tween 20, following the addition of 2 lg rBP53 and incubation for 2 h at 37 °C. After three times wash, 1:1000 rabbit anti-rBP53 polyclonal antibody was added and incubated for 2 h at 37 °C. After another wash, plates were incubated with HRP-labeled goat anti-rabbit IgG antibody for 2 h at 37 °C. Finally, the reaction was visualized using the HRP substrate O-phenylenediamine, and the reaction was stopped by the addition of 2 M sulfuric acid (H2SO4) solution. Absorbance was immediately read at 492 nm using a Varioskan Flash (Thermo Fisher Scientific Inc., USA) plate reader. 1.6. Co-localization analysis for VP37 and BP53 To determine whether VP37 and BP53 could interact at the cellular level, an immunofluorescence co-localization assay was performed. This experiment was carried out as described previously with some modifications (George and Dhar, 2010). Hemolymph was withdrawn from the WSSV-free shrimp Litopeneaus vannamei, and hemocytes were collected by centrifugation at 500g for 5 min with anticoagulants (27 mM Sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0), then pellets were suspended in 2 L15 medium (GIBCO, USA) with 19% (vol/vol) fetal calf serum (FCS) and 1% (vol/vol) penicillin and streptomycin. After incubation at 28 °C for 1 h to allow cells to attach, hemocytes, either be permeabilized using 0.5% TritonX-100 or be fixed but not permeabilized, were incubated at 28 °C for 2 h with FITC-conjugated rabbit anti-rBP53 polyclonal antibody (Sigma Inc., USA) and Alexa Flour 647-conjugated rVP37 (Life Technologies, USA). FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA staining experiments were also performed as negative control. Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Roche Inc., USA). Cells were observed using confocal laser scanning microscopy (Nikon Inc., Japan). Co-localization was analyzed graphically in a scatterplot using specialized software (NISElements AR 3.0). 2. Results
Primer name
Primer sequence
Amplified fragment (bp)
2.1. Expression and purification of rVP37
pBAD/gIII A-VP37f pBAD/gIII A-VP37r 16SrDNAf 16SrDNAr
50 -ATGCCATAGCATTTTTATCC-30 50 -GATTTAATCTGTATCAGG-30 50 -TGAGTAATGCCTGGGAAATTGC-30 50 -ATCGTCGCCTTGGTGAGC-30
1000
The VP37 gene encoding 281 amino acids with a theoretical molecular mass of 37 kDa was inserted into the pBAD/gIIIA plasmid and expressed in E. coli TOP10 as a fusion protein. PCR amplification of pBAD/gIIIA-vp37 yielded a 1000 bp DNA fragment with
304
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the expected 838 bp vp37 sequence, and there was no specific fragment in the ‘‘not-induced” E. coli TOP10. Additionally, the 16SrDNA fragment (304 bp) was identified by all bacteria (Fig. 1). rVP37 was purified by using affinity chromatography system, an absorption peak was observed by measurement at UV 280 nm (Fig. 2). By coomassie-blue stained SDS–PAGE analysis, an expected molecular mass of 37 kDa was detected in the total proteins, which was successfully purified from the precipitated bacterial lysates of induced E. coli (Fig. 3, lane 5) compared with noninduced bacteria (Fig. 3, lanes 1–4) 2.2. BP53 binds to three WSSV proteins To identify the BP53-binding proteins in WSSV, far-western blotting was carried out. WSSV proteins were separated by SDS– PAGE and transferred to a PVDF membrane, followed by the application of purified rBP53 as a probe to react with viral proteins. Three bands at 28, 37, and >50 kDa were recognized by BP53 (Fig. 4, Lane 1). The proteins at 28 and 37 kDa were identified as VP28 and VP37, two of the envelope proteins of WSSV, which were recognized by their polyclonal antibody respectively (Fig. 4, Lane 2–3). 2.3. Determination of the binding capability between VP37 and BP53 In the binding assay, plates coated with 5% fat-free milk served as negative controls. Results were expressed as an absorbance ratio of samples over negative controls (i.e., P/N). P/N P 2.1 was designated as positive. We found that rBP53 had a significant binding capability for rVP37 compared with a negative control. Measured P/N values revealed that the specific binding between rBP53 to rVP37 reached almost 80% of the binding capability between rBP53 to WSSV (Fig. 5). 2.4. WSSV-VP37 colocalized with BP53 on shrimp hemocytes An interaction between WSSV-VP37 and BP53 was identified using the far-western and ELISA binding assay at the molecular level, and an immunofluorescence co-localization assay indicated that this occurred at the cellular level. Finally, we demonstrate extensive co-localization of bound rVP37 and BP53 on the cell sur-
Fig. 1. Amplification of pBAD/gIIIA-VP37 and 16SrDNA in E. coli by PCR. Lane 1–3, amplification of pBAD/gIIIA-VP37 in induced bacteria; Lane 4, amplification of pBAD/gIIIA-VP37 in non-induced bacteria; Lane 5–8, amplification of 16S rDNA; Lane M, DNA marker (DL2000, 100–2000 bp).
face using confocal microscopy with Alexa Flour 647-labeled rVP37 and FITC-conjugated rabbit anti-rBP53 polyclonal antibody. Fig. 6 (columns 2 and 3) show BP53 and WSSV-VP37 staining, respectively, and Fig. 6 (column 4) shows an overlay of these three images. Images in Fig. 6 (column 4) clearly show an overlapping distribution (yellow signal) of these two proteins. Staining for rVP37 (red signal) appears around the punctate structures that contain BP53 (green signal) along the periphery of these cells. As observed in the overlayed images (Fig. 6, column 4), no fluorescent signals for BP53 appeared in a cluster (Fig. 6, row A–C) or in uneven dots (Fig. 6, row D and E) in the non-permeabilized cells, and all signal encircled by that of bound rVP37 occurred in a punctual and uneven fashion (Fig. 6, column 4). The control staining experiments using FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA did not yield any signal on hemocytes, which excluded the possibility of non-specific interactions (Fig. 6 row F). Quantitative associations between the fluorescent signals for co-localization are presented graphically in a scatterplot, which was analyzed using specialized software (NIS-Elements AR 3.0). A linear relationship indicating an interaction was observed when the pixel intensity of red color (Alexa Flour 647) was plotted against that of green color (FITC), as shown in Fig. 6G–H). Imaging data are shown in Fig. 6A–E to evaluate co-localization. Our results show that the Pearson’s correlation coefficients reached 0.67 ± 0.05 and the Mander overlap reached 0.70 ± 0.05, suggesting that there was a strong co-relationship exists between the localization of BP53 and that of bound rVP37. A comparison of Mander’s overlap coefficients (K1 and K2) and co-localization coefficients (C1 and C2), provided a quantitative assessment of co-localization between BP53 and rVP37 (Table 2).
3. Discussion In the last five years, increasing numbers of virus-binding proteins (VBPs) have been identified in shrimp, which has provided a better understanding of how host shrimp respond to viral infections and of the shrimp innate immune response (Sritunyalucksana et al., 2012). These findings suggest that the process of WSSV infection is extremely complex. In our previous study, ATPsyn b subunit (named as BP53) was identified on the shrimp cell surface of hemocytes and gill epithelia, which plays an important role in WSSV infection by acting as a receptor for WSSV (Liang et al., 2010; G.F. Liang et al., 2015; Y. Liang et al., 2015). However, the viral proteins that could interact with BP53 have remained unknown. Herein, we identified three proteins from WSSV that can interact with BP53, including two known envelope proteins, VP28 and VP37. Lipid rafts are known to be involved in efficient virus infection (Yang et al., 2015; Hawkes et al., 2015; Zhu et al., 2015), while the ATP synthase complex (a and b) may be located in plasma membrane lipid rafts (Bae et al., 2004). Based on the aforementioned studies, our data led to the hypothesis that WSSV can enter cells via receptor BP53-mediated endocytosis. This model is supported by the findings of Huang et al., which suggested that WSSV utilizes caveolae-mediated endocytosis to enter shrimp cells, because of the inhibition of fluorescent dyes that labeled virion entry into hemocytes by an endocytic inhibitor, Methyl-bcyclodextrin (MbCD) (Huang et al., 2013). Our studies of viral proteins that can interact with BP53 advance our understanding of the role of BP53 in the process of infection by WSSV. VP37, an envelope protein of WSSV, has been identified as a viral attachment protein of WSSV that can interact with shrimp cells (Liang et al., 2005; Liu et al., 2009). Recently, VP37-binding protein was reported to be BP53 in the shrimp L. vannamei by a virus overlay protein binding and GST pull-down assays (Zhan et al., 2013). Our findings also supported binding activity between
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Fig. 2. Purification of rVP37 using the affinity chromatography system. The arrow indicates the absorption peak of purified expressed protein that measured at UV 280 nm.
VP37 and BP53, indicating that VP37 is a binding protein for BP53. This finding is also supported by our co-localization analysis, which is a frequently used technique to determine whether two molecules associate with the same binding sites (Dunn et al., 2011). We found that VP37 was co-localized with BP53, and shared the same subcellular location on the external surface of shrimp cells. Co-localization imaging can provide visual evidence to support the interactions of two or more molecules, which is governed by specific biological processes, such as compartmentalization and binding (Zinchuk and Grossenbacher-Zinchuk, 2009). By quantitatively calculating the co-localization between BP53 and rVP37, the Manders’ overlap coefficients reached 0.7 and the co-localization coefficients C1 was 0.94, which represent a true and high degree of co-localization between BP53 and VP37 and better established the co-interaction between BP53 and VP37. Using an ELISA binding assay, we detected specific binding between rBP53 to rVP37 that
Fig. 4. Identification of BP53-binding proteins from WSSV. Far-western blotting of rBP53 to the WSSV proteins, and HRP-conjugated goat anti-rabbit IgG was used as a secondary antibody. Lane 1, incubation with rBP53 and rabbit anti-rBP53-antibody; Lane 2, incubation with rabbit anti-rVP37-antibody; Lane 3, incubation with rabbit anti-rVP28-antibody. Arrows indicate the binding proteins.
6 5
P/N value
Fig. 3. Coomassie-blue stained SDS–PAGE analysis of bacterial lysates isolated from E. coli and purification of rVP37. Lane 1, bacterial lysates isolated from not-induced E. coli; Lane 2 to 4, total protein, supernatant, and precipitate of bacterial lysates isolated from induced E. coli; 5 respectively; Lane 5, purified rVP37 protein; Lane M, protein marker (14–116 kDa). Arrow indicates rVP37, which was detected in the total proteins and precipitation of bacterial lysates isolated from induced E. coli.
4 3 2 1 0 skim milk
VP37
WSSV
Fig. 5. Binding capability analysis between rBP53, rVP37 and WSSV by binding assay. Absorbance measured at 492 nm. Means ± standard deviations (n = 3) of P/N value are reported.
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Fig. 6. A panel of confocal microscope images showed co-localization of bound rVP37 and BP53 on hemocytes. Representative images are shown (row A–E). FITC-conjugated rabbit anti-rBP53 polyclonal antibody was used to visualize BP53, which is shown in green color (column 2). Alexa Flour 647-conjugated rVP37 bound on hemocytes is shown in red color (column 3). Cell nuclei were stained with DAPI in blue color (column 1). Corresponding merged images are shown in column 4. FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA were used as staining controls (row F). A linear relationship indicating an interaction was observed when the pixel intensity of red color (Alexa Flour 647) was plotted against that of green color (FITC). The result shown in G is from the image shown in (F) (control), and the result shown in H is from images in (A–E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Quantitative calculation analysis on images with co-localization. Pearson’s correlation
0.67 ± 0.05
Mander’s overlap
0.70 ± 0.05
Mander’s overlap coefficients
Colocalization coefficients
K1
K2
C1
C2
0.54 ± 0.15
1.04 ± 0.37
0.94 ± 0.05
0.66 ± 0.17
accounted for almost 80% of the binding capability between rBP53 and WSSV based on absorbance data measured at 492 nm. Perhaps, the remaining 20% of binding capability of WSSV and rBP53 contributed by interacting with the other two binding proteins, VP28 and the >50 kDa protein. In a separate study, we found that both VP37 and shrimp astakine – an invertebrate cytokine that functions in promoting hematopoiesis in crustaceans – use BP53 on the target cell surface as a receptor, and they both likely could competitively bind to BP53 (G.F. Liang et al., 2015). This finding indicates that VP37 might play a key role in mediating or facilitat-
ing WSSV invasion into host cells by binding to the cell surface and then going on to impair the shrimp innate immune system.
4. Conclusions Together, our findings provide evidence that support the role of BP53 as a receptor for WSSV by interacting with three viral proteins. Furthermore, our study identified an interaction between BP53 and VP37 at the molecular and cellular levels, and revealed
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the important role of VP37 in mediating WSSV entry because of its high binding affinity for BP53. Our findings also likely indicated that the process of WSSV infection is likely to be complex because several binding proteins were identified. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant Numbers: 31101934 and 31302233), the Special Scientific Research Funds for Central Non-Profit Institutes, Yellow Sea Fisheries Research Institute (Grant Number: 20603022013009), the National Basic Research Program (973 Program) of China (Grant No. 2012CB114400), and the special foundation under the Construction Program for ‘‘Taishan Scholarship” of Shandong Province of China. References Bae, T.J., Kim, M.S., Kim, J.W., Kim, B.W., Choo, H.J., Lee, J.W., Kim, K.B., Lee, C.S., Kim, J.H., Chang, S.Y., Kang, C.Y., Lee, S.W., Ko, Y.G., 2004. Lipid raft proteome reveals ATP synthase complex in the cell surface. Proteomics 4 (11), 3536–3548. Boyle, J.F., Weismiller, D.G., Holemes, K.V., 1987. Genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues. J. Virol. 61, 185–193. Compton, T., Nowlin, D.M., Cooper, N.R., 1993. Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193 (1), 834–841. Dunn, K.W., Kamocka, M.M., McDonald, J.H., 2011. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300 (4), C723–C742. George, S.K., Dhar, A.K., 2010. An improved method of cell culture system from eyestalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei. Vitro Cell. Dev. Biol. Anim. 46, 801–810. Hawkes, D., Jones, K.L., Smyth, R.P., Pereira, C.F., Bittman, R., Jaworowski, A., Mak, J., 2015. Properties of HIV-1 associated cholesterol in addition to raft formation are important for virus infection. Virus Res. 210, 18–21. Huang, Z.J., Kang, S.T., Leu, J.H., Chen, L.L., 2013. Endocytic pathway is indicated for white spot syndrome virus (WSSV) entry in shrimp. Fish Shellfish Immunol. 35 (3), 707–715. Liang, Y., Huang, J., Song, X.L., Zhang, P.J., Xu, H.S., 2005. Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes. Diseases Aquatic Organ. 66, 81–85. Liang, Y., Cheng, J.J., Yang, B., Huang, J., 2010. The role of F1 ATP synthase beta subunit in WSSV infection in the shrimp, Litopenaeus vannamei. Virol. J. 7, 144. Liang, G.F., Liang, Y., Xue, Q.G., Lu, J.F., Cheng, J.J., Huang, J., 2015a. Astakine LvAST binds to the beta subunit of F1-ATP synthase and likely plays a role in white shrimp Litopeneaus vannamei defense against white spot syndrome virus. Fish Shellfish Immunol. 43, 75–81.
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Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus Chen Li, Xiao-Xiao Gao, Jie Huang, Yan Liang ⇑ Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
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Article history: Received 29 September 2015 Revised 9 January 2016 Accepted 12 January 2016 Available online 13 January 2016 Keywords: WSSV Shrimp F1 ATP synthase b subunit BP53 VP37 Interaction
a b s t r a c t The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase b subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson’s correlation coefficients reached to 0.67 ± 0.05 and the Mander’s overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. Ó 2016 Elsevier Inc. All rights reserved.
0. Introduction For a virus to initiate its life cycle, specific binding must occur between viral attachment proteins (VAPs) and cellular receptors on host cells, which mediates virus entry and triggers subsequent viral infection (Boyle et al., 1987). Associations between VAPs and host receptor molecules can occur via high- or low-affinity interactions, and are thought to be important for cell fusion and viral replication (Staunton et al., 1989; Compton et al., 1993). The shrimp farming industry is affected by several viral diseases that cause billions of dollars of economic losses annually (Lightner, 1996). White spot syndrome virus (WSSV, family Nimaviridae, genus Whispovirus), an enveloped, double-stranded DNA virus, containing 180 putative open reading frames (ORFs) in its complete genome sequence (Tsai et al., 2000; Van et al., 2001; Yang et al., 2001), is a severe viral pathogen (Lightner, 1996). Over 50 structural and several non-structural protein genes of WSSV have ⇑ Corresponding author at: No. 106 Nanjing Road, Qingdao 266071, China. E-mail address: [email protected] (Y. Liang). http://dx.doi.org/10.1016/j.jip.2016.01.006 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
been characterized, including VP26, VP28, VP68, VP187, and VP466 that have been reported to be significantly involved in WSSV infection (Xie and Yang, 2005; Wu et al., 2005; Sritunyalucksana et al., 2006; Ye et al., 2012). Previously, we suggested that the envelope protein WSSV-VP37 is a viral attachment protein and can interact with shrimp cells (Liang et al., 2005; Liu et al., 2006; Liu et al., 2009). Virus-host cell interactions play an important role in WSSV infection, as summarized by Sritunyalucksana et al. (2012). Using a virus overlay protein binding assay (VOPBA) with WSSV and in vivo neutralization tests, we found that shrimp F1 ATP synthase b subunit (ATPsyn b, termed BP53) plays a role in WSSV infection (Liang et al., 2010). Further studies of BP53 revealed its location on the cell surface of shrimp hemocytes and gill epithelial cells, which meet the basic requirements for it to be a viral receptor (Zhan et al., 2013). In our previous study, we showed evidence that ATP synthesis is active on the shrimp cell surface and can be suppressed by WSSV infection (Y. Liang et al., 2015). As ATP synthase b – a part of the F1Fo ATP synthase complex – can serve as a receptor for WSSV, our findings support the hypothesis that WSSV disturbs host energy
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metabolism by interacting with host BP53 molecules (Y. Liang et al., 2015). Recently, BP53 was reported to be a VP37-binding protein (Zhan et al., 2013). However it’s unclear how many viral proteins, excluding VP37, could interact with BP53. There also lacks detailed description on the relationship between shrimp BP53 and WSSV-VP37. This study aimed to identify and assess interactions between BP53 and its viral binding proteins by both qualitative and quantitative analyses. Investigating the relationship between shrimp BP53 and viral binding proteins might represent an interesting way to better understand the molecular pathways involved in WSSV infection.
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to react with viral proteins, and was incubated with the eluted products at 37 °C for 2 h. After three washes with PBS containing 0.05% Tween 20, membranes were incubated with 1:5000 rabbit anti-rBP53 polyclonal antibody at 37 °C for 2 h. The WSSV viral proteins on the other membrane were incubated with either 1:5000 rabbit anti-rVP37 polyclonal antibody or rabbit antirVP28 polyclonal antibody at 37 °C for 2 h. After three times wash, the signal was detected using a HRP-conjugated goat anti-rabbit IgG secondary antibody. After thorough washing, the color was developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., USA). 1.5. Determination of binding capability between VP37 and BP53
1. Materials and methods 1.1. Virus source Intact WSSV viral particles from infected crayfish tissues were purified by differential centrifugation as described by Xie et al. (2005). Viral protein concentrations were measured using the Bradford method (Stoscheck, 1990) and proteins were stored at 80 °C. 1.2. Recombinant VP37 and BP53 Recombinant VP37 (rVP37) and recombinant BP53 (rBP53) were produced in an Escherichia coli expression system, as reported by Liu et al. (2009) and Liang et al. (2010), respectively. In brief, TOP10s encoding recombinant plasmid pBAD/gIII A-VP37 were detected by PCR, and 16S rDNA was used as a reference gene. Primers designed using Primer Premier 5.0 showed in Table 1. The rVP37 was purified using HisTrap HF resin and GE AKTA Explorer 100 (GE Inc., USA) according to the manufacturer’s instructions under denatured conditions, and then was renatured by successive for 12 h incubations with 6, 4, 2, and 0 M Guanidine–HCl in buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 25 mM dithiothreitol, 0.1% Tween-20, and 10% glycerol, pH 7.5) at 4 °C. Fractions were collected using the Centrifugal Filters (Millipore Inc., USA) and then analyzed by SDS–PAGE. 1.3. Polyclonal antibodies Rabbit polyclonal antibodies against VP37 and VP28 were custom prepared by GenScript (Nanjing Co. in China) by immunizing New Zealand white rabbits with either purified antigenic protein rVP37 or rVP28. The rabbit polyclonal antibody against BP53 was prepared as reported by Y. Liang et al. (2015). Antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). 1.4. Determination of binding proteins for BP53 Far-western blotting assays were carried out to identify binding proteins for BP53. The WSSV proteins were separated by 12% SDS– PAGE and transferred to a membrane. After blocking with 5% skim milk in PBS at 37 °C for 1 h, purified rBP53 was applied as a probe Table 1 Primers for pBAD/gIII A-VP37 and 16SrDNA.
To determine the binding capability of rVP37 and WSSV viral proteins with rBP53, an ELISA binding assay was done. Along with a similar amount of protein (30 lg) in skim milk, rVP37 and WSSV viral proteins were coated in flat-bottomed 96-well plates (Corning, China) at 4 °C overnight and then were blocked with 5% nonfat milk in PBS buffer for 2 h at 37 °C. Plates were washed three times with PBS buffer that contained 0.05% Tween 20, following the addition of 2 lg rBP53 and incubation for 2 h at 37 °C. After three times wash, 1:1000 rabbit anti-rBP53 polyclonal antibody was added and incubated for 2 h at 37 °C. After another wash, plates were incubated with HRP-labeled goat anti-rabbit IgG antibody for 2 h at 37 °C. Finally, the reaction was visualized using the HRP substrate O-phenylenediamine, and the reaction was stopped by the addition of 2 M sulfuric acid (H2SO4) solution. Absorbance was immediately read at 492 nm using a Varioskan Flash (Thermo Fisher Scientific Inc., USA) plate reader. 1.6. Co-localization analysis for VP37 and BP53 To determine whether VP37 and BP53 could interact at the cellular level, an immunofluorescence co-localization assay was performed. This experiment was carried out as described previously with some modifications (George and Dhar, 2010). Hemolymph was withdrawn from the WSSV-free shrimp Litopeneaus vannamei, and hemocytes were collected by centrifugation at 500g for 5 min with anticoagulants (27 mM Sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0), then pellets were suspended in 2 L15 medium (GIBCO, USA) with 19% (vol/vol) fetal calf serum (FCS) and 1% (vol/vol) penicillin and streptomycin. After incubation at 28 °C for 1 h to allow cells to attach, hemocytes, either be permeabilized using 0.5% TritonX-100 or be fixed but not permeabilized, were incubated at 28 °C for 2 h with FITC-conjugated rabbit anti-rBP53 polyclonal antibody (Sigma Inc., USA) and Alexa Flour 647-conjugated rVP37 (Life Technologies, USA). FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA staining experiments were also performed as negative control. Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Roche Inc., USA). Cells were observed using confocal laser scanning microscopy (Nikon Inc., Japan). Co-localization was analyzed graphically in a scatterplot using specialized software (NISElements AR 3.0). 2. Results
Primer name
Primer sequence
Amplified fragment (bp)
2.1. Expression and purification of rVP37
pBAD/gIII A-VP37f pBAD/gIII A-VP37r 16SrDNAf 16SrDNAr
50 -ATGCCATAGCATTTTTATCC-30 50 -GATTTAATCTGTATCAGG-30 50 -TGAGTAATGCCTGGGAAATTGC-30 50 -ATCGTCGCCTTGGTGAGC-30
1000
The VP37 gene encoding 281 amino acids with a theoretical molecular mass of 37 kDa was inserted into the pBAD/gIIIA plasmid and expressed in E. coli TOP10 as a fusion protein. PCR amplification of pBAD/gIIIA-vp37 yielded a 1000 bp DNA fragment with
304
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the expected 838 bp vp37 sequence, and there was no specific fragment in the ‘‘not-induced” E. coli TOP10. Additionally, the 16SrDNA fragment (304 bp) was identified by all bacteria (Fig. 1). rVP37 was purified by using affinity chromatography system, an absorption peak was observed by measurement at UV 280 nm (Fig. 2). By coomassie-blue stained SDS–PAGE analysis, an expected molecular mass of 37 kDa was detected in the total proteins, which was successfully purified from the precipitated bacterial lysates of induced E. coli (Fig. 3, lane 5) compared with noninduced bacteria (Fig. 3, lanes 1–4) 2.2. BP53 binds to three WSSV proteins To identify the BP53-binding proteins in WSSV, far-western blotting was carried out. WSSV proteins were separated by SDS– PAGE and transferred to a PVDF membrane, followed by the application of purified rBP53 as a probe to react with viral proteins. Three bands at 28, 37, and >50 kDa were recognized by BP53 (Fig. 4, Lane 1). The proteins at 28 and 37 kDa were identified as VP28 and VP37, two of the envelope proteins of WSSV, which were recognized by their polyclonal antibody respectively (Fig. 4, Lane 2–3). 2.3. Determination of the binding capability between VP37 and BP53 In the binding assay, plates coated with 5% fat-free milk served as negative controls. Results were expressed as an absorbance ratio of samples over negative controls (i.e., P/N). P/N P 2.1 was designated as positive. We found that rBP53 had a significant binding capability for rVP37 compared with a negative control. Measured P/N values revealed that the specific binding between rBP53 to rVP37 reached almost 80% of the binding capability between rBP53 to WSSV (Fig. 5). 2.4. WSSV-VP37 colocalized with BP53 on shrimp hemocytes An interaction between WSSV-VP37 and BP53 was identified using the far-western and ELISA binding assay at the molecular level, and an immunofluorescence co-localization assay indicated that this occurred at the cellular level. Finally, we demonstrate extensive co-localization of bound rVP37 and BP53 on the cell sur-
Fig. 1. Amplification of pBAD/gIIIA-VP37 and 16SrDNA in E. coli by PCR. Lane 1–3, amplification of pBAD/gIIIA-VP37 in induced bacteria; Lane 4, amplification of pBAD/gIIIA-VP37 in non-induced bacteria; Lane 5–8, amplification of 16S rDNA; Lane M, DNA marker (DL2000, 100–2000 bp).
face using confocal microscopy with Alexa Flour 647-labeled rVP37 and FITC-conjugated rabbit anti-rBP53 polyclonal antibody. Fig. 6 (columns 2 and 3) show BP53 and WSSV-VP37 staining, respectively, and Fig. 6 (column 4) shows an overlay of these three images. Images in Fig. 6 (column 4) clearly show an overlapping distribution (yellow signal) of these two proteins. Staining for rVP37 (red signal) appears around the punctate structures that contain BP53 (green signal) along the periphery of these cells. As observed in the overlayed images (Fig. 6, column 4), no fluorescent signals for BP53 appeared in a cluster (Fig. 6, row A–C) or in uneven dots (Fig. 6, row D and E) in the non-permeabilized cells, and all signal encircled by that of bound rVP37 occurred in a punctual and uneven fashion (Fig. 6, column 4). The control staining experiments using FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA did not yield any signal on hemocytes, which excluded the possibility of non-specific interactions (Fig. 6 row F). Quantitative associations between the fluorescent signals for co-localization are presented graphically in a scatterplot, which was analyzed using specialized software (NIS-Elements AR 3.0). A linear relationship indicating an interaction was observed when the pixel intensity of red color (Alexa Flour 647) was plotted against that of green color (FITC), as shown in Fig. 6G–H). Imaging data are shown in Fig. 6A–E to evaluate co-localization. Our results show that the Pearson’s correlation coefficients reached 0.67 ± 0.05 and the Mander overlap reached 0.70 ± 0.05, suggesting that there was a strong co-relationship exists between the localization of BP53 and that of bound rVP37. A comparison of Mander’s overlap coefficients (K1 and K2) and co-localization coefficients (C1 and C2), provided a quantitative assessment of co-localization between BP53 and rVP37 (Table 2).
3. Discussion In the last five years, increasing numbers of virus-binding proteins (VBPs) have been identified in shrimp, which has provided a better understanding of how host shrimp respond to viral infections and of the shrimp innate immune response (Sritunyalucksana et al., 2012). These findings suggest that the process of WSSV infection is extremely complex. In our previous study, ATPsyn b subunit (named as BP53) was identified on the shrimp cell surface of hemocytes and gill epithelia, which plays an important role in WSSV infection by acting as a receptor for WSSV (Liang et al., 2010; G.F. Liang et al., 2015; Y. Liang et al., 2015). However, the viral proteins that could interact with BP53 have remained unknown. Herein, we identified three proteins from WSSV that can interact with BP53, including two known envelope proteins, VP28 and VP37. Lipid rafts are known to be involved in efficient virus infection (Yang et al., 2015; Hawkes et al., 2015; Zhu et al., 2015), while the ATP synthase complex (a and b) may be located in plasma membrane lipid rafts (Bae et al., 2004). Based on the aforementioned studies, our data led to the hypothesis that WSSV can enter cells via receptor BP53-mediated endocytosis. This model is supported by the findings of Huang et al., which suggested that WSSV utilizes caveolae-mediated endocytosis to enter shrimp cells, because of the inhibition of fluorescent dyes that labeled virion entry into hemocytes by an endocytic inhibitor, Methyl-bcyclodextrin (MbCD) (Huang et al., 2013). Our studies of viral proteins that can interact with BP53 advance our understanding of the role of BP53 in the process of infection by WSSV. VP37, an envelope protein of WSSV, has been identified as a viral attachment protein of WSSV that can interact with shrimp cells (Liang et al., 2005; Liu et al., 2009). Recently, VP37-binding protein was reported to be BP53 in the shrimp L. vannamei by a virus overlay protein binding and GST pull-down assays (Zhan et al., 2013). Our findings also supported binding activity between
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Fig. 2. Purification of rVP37 using the affinity chromatography system. The arrow indicates the absorption peak of purified expressed protein that measured at UV 280 nm.
VP37 and BP53, indicating that VP37 is a binding protein for BP53. This finding is also supported by our co-localization analysis, which is a frequently used technique to determine whether two molecules associate with the same binding sites (Dunn et al., 2011). We found that VP37 was co-localized with BP53, and shared the same subcellular location on the external surface of shrimp cells. Co-localization imaging can provide visual evidence to support the interactions of two or more molecules, which is governed by specific biological processes, such as compartmentalization and binding (Zinchuk and Grossenbacher-Zinchuk, 2009). By quantitatively calculating the co-localization between BP53 and rVP37, the Manders’ overlap coefficients reached 0.7 and the co-localization coefficients C1 was 0.94, which represent a true and high degree of co-localization between BP53 and VP37 and better established the co-interaction between BP53 and VP37. Using an ELISA binding assay, we detected specific binding between rBP53 to rVP37 that
Fig. 4. Identification of BP53-binding proteins from WSSV. Far-western blotting of rBP53 to the WSSV proteins, and HRP-conjugated goat anti-rabbit IgG was used as a secondary antibody. Lane 1, incubation with rBP53 and rabbit anti-rBP53-antibody; Lane 2, incubation with rabbit anti-rVP37-antibody; Lane 3, incubation with rabbit anti-rVP28-antibody. Arrows indicate the binding proteins.
6 5
P/N value
Fig. 3. Coomassie-blue stained SDS–PAGE analysis of bacterial lysates isolated from E. coli and purification of rVP37. Lane 1, bacterial lysates isolated from not-induced E. coli; Lane 2 to 4, total protein, supernatant, and precipitate of bacterial lysates isolated from induced E. coli; 5 respectively; Lane 5, purified rVP37 protein; Lane M, protein marker (14–116 kDa). Arrow indicates rVP37, which was detected in the total proteins and precipitation of bacterial lysates isolated from induced E. coli.
4 3 2 1 0 skim milk
VP37
WSSV
Fig. 5. Binding capability analysis between rBP53, rVP37 and WSSV by binding assay. Absorbance measured at 492 nm. Means ± standard deviations (n = 3) of P/N value are reported.
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Fig. 6. A panel of confocal microscope images showed co-localization of bound rVP37 and BP53 on hemocytes. Representative images are shown (row A–E). FITC-conjugated rabbit anti-rBP53 polyclonal antibody was used to visualize BP53, which is shown in green color (column 2). Alexa Flour 647-conjugated rVP37 bound on hemocytes is shown in red color (column 3). Cell nuclei were stained with DAPI in blue color (column 1). Corresponding merged images are shown in column 4. FITC-conjugated pre-immune serum and Alexa Flour 647-labeled BSA were used as staining controls (row F). A linear relationship indicating an interaction was observed when the pixel intensity of red color (Alexa Flour 647) was plotted against that of green color (FITC). The result shown in G is from the image shown in (F) (control), and the result shown in H is from images in (A–E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Quantitative calculation analysis on images with co-localization. Pearson’s correlation
0.67 ± 0.05
Mander’s overlap
0.70 ± 0.05
Mander’s overlap coefficients
Colocalization coefficients
K1
K2
C1
C2
0.54 ± 0.15
1.04 ± 0.37
0.94 ± 0.05
0.66 ± 0.17
accounted for almost 80% of the binding capability between rBP53 and WSSV based on absorbance data measured at 492 nm. Perhaps, the remaining 20% of binding capability of WSSV and rBP53 contributed by interacting with the other two binding proteins, VP28 and the >50 kDa protein. In a separate study, we found that both VP37 and shrimp astakine – an invertebrate cytokine that functions in promoting hematopoiesis in crustaceans – use BP53 on the target cell surface as a receptor, and they both likely could competitively bind to BP53 (G.F. Liang et al., 2015). This finding indicates that VP37 might play a key role in mediating or facilitat-
ing WSSV invasion into host cells by binding to the cell surface and then going on to impair the shrimp innate immune system.
4. Conclusions Together, our findings provide evidence that support the role of BP53 as a receptor for WSSV by interacting with three viral proteins. Furthermore, our study identified an interaction between BP53 and VP37 at the molecular and cellular levels, and revealed
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